1. Field of the Invention
This invention relates to biologically active polypeptides and their production from natural or synthetic sorces, oligopeptides derived from said polypeptides, and compositions methods for usefully applying the biological activity associated with the polypeptides and oligopeptides derived therefrom in the health sciences field. More particularly, this invention is directed to a general polypeptide structure which defines proteins having cell growth promoting activity and a novel class of transforming growth factor (TGF) polypeptides which have the property of reversibly conferring the transformed phenotype on normal cells in vitro and thus appear to be proximate effectors of the malignant phenotype. In another particular aspect, this invention is directed to antigenic oligopeptides derived from the TGF polypeptides and to antibodies raised to the antigenic oligopeptides and the associated TGF polypeptides which are useful in the detection and treatment of malignancies. In a further particular aspect, this invention is directed to a class of oligopeptides which have the ability to bind to cellular growth factor receptors and thus to potentially interfere with transformation of certain cell lines into a cancerous state. In a still further particular aspect, this invention is directed to a process for isolating TGF polypeptides in homogeneous form from both transformed human and murine cell lines and body fluids and to the homogeneous TGF polypeptides so obtained. Other particular aspects of this invention are directed to compositions and methods for detection and treatment of cancer and other proliferative diseases and for cell growth associated treatment, for example, wound healing and ulcer treatment. Furthermore, another aspect of this invention is in the treatment and detection of bone-loss diseases, such as osteoporosis and hypercalcemia.
A number of polypeptide hormone and hormone-like growth factors have been found in tissue fluids and their relationship in the control of normal cellular growth or mitosis has been established. These mitogenic polypeptide growth factors include insulin, insulin-like growth factors, platelet-derived growth factor, nerve growth factor, fibroblast growth factor and epidermal growth factor (EGF). At least some of these known growth factors have an effect on the growth of transformed cells, however, on the basis of in vitro tests, it appears that transformed cells require less of these known growth factors for optimal growth and multiplication than do normal cells. In particular, it has been shown, in experiments in cell culture, that the addition of exogenous growth factors such as insulin and EGF can cause normal cells to mimic certain changes in cellular properties that are analogous to transformation; however, they are unable to produce all of the changes associated with the transformed phenotype, e.g., see Sporn et al. (1980) The New Eng. J. of Med. 15, pp. 878-880.
Recently, new types of polypeptide growth factors designated as transforming growth factors or TGFs have been found in certain human and animal carcinoma and sarcoma cells which possess a greater complement of the properties apparently essential to phenotypic transformation (Roberts et al. (1980) Proc. Natl. Acad. Sci. USA 77, pp. 3494-3498 and Todaro et al. (1980) Proc. Natl. Acad. Sci. USA 77, pp. 5258-5262). The TGF polypeptides as a class are characterized by the changes which they cause when applied to untransformed, non-neoplastic indicator cells growing in culture. These changes include (a) loss of density-dependent inhibition of cell growth in monolayer culture, (b) overgrowth of cells in monolayer culture, (c) change in cellular shape, with the result that the indicator cells assume the neoplastic phenotype, and (d) acquisition of anchorage-independence, with the resultant ability to grow in soft agar. The property of anchorage-independent growth of cells in culture has a particularly high correlation with neoplastic growth in vivo. At least certain of the TGF polypeptides show some relationship with EGF in that they are both heat-stable, acid-stable peptides sensitive to reducing agents and proteases and they appear to specifically interact with, and produce biological effects through, cellular membrane EGF receptors, TGF competing with EGF for binding to the cellular EGF receptor. However, TGF is distinguishable from EGF in several important respects. In particular, EGF does not induce anchorage-independent growth of cells in culture nor do antibodies to EGF detect TGF in either radioimmunoassay or immunoprecipitation tests. Further, EGF has only a slight effect on the phenotype of cultured cells, whereas TGF produces a more pronounced phenotypic alteration in cultured cells and confers on them the ability to behave as transformed cells. Interestingly, the transformation produced by TGF is not permanent but reversible in the absence of TGF and there is no evidence that TGF acts as a complete carcinogen itself. (Todaro et al. (1981) J. of Supramolecular Structure and Cell Biochem. 15, pp. 287-301).
Thus, TGF polypeptides are a unique class of proteins distinguishable from other growth factors such as EGF from the standpoint of both biological properties and chemical structure. These TGFs, in turn, possess a variety of properties of value or potential value in the health sciences field including potent, but reversible, cell growth or mitogenic properties which find use in cell repair including, for example, wound healing and ulcer therapy. Additionally, the production of TGF polypeptides, or elevated levels of production, are characteristic of, if not essential to, the morphologic transformation of certain cell lines in both human and murine tissue and/or fluids; therefore, the TGF polypeptides or antigenic fragments thereof are of value in differentiating normal cells from tumor cells and antibodies raised thereto have application in both the diagnosis and treatment of malignancies. Further, realization that certain TGF polypeptides specifically interact with and produce their biological effects through cellular membrane EGF receptors raises the possibility, once the basic TGF polypeptide structure is determined, of correlating its structure with the structure of EGF to develop oligopeptides having chemical characteristics to allow binding to the EGF receptors without concomitant phenotypic transformation of the cell. Oligopeptides having this characteristic EGF receptor binding ability find application in treatment of malignancies, since the oligopeptide will interfere or compete with TGF for available receptor sites and thereby interrupt the expression of the transformed properties of the cell.
With the present invention, a method has been developed to obtain TGF polypeptides in sufficient quantity and purity to allow complete structure determination and as a result of this determination and other observations, including the finding of substantial homology between human and murine TGFs, a basic peptide structure has been discovered which has broad application in cell mitosis and the cell growth related field of use. Further, homogeneous TGF polypeptides are obtained having application in both the cell growth field and in the detection and treatment of cancer and other proliferative diseases.
TGF polypeptides, oligopeptides and antibodies raised to these polypeptides also have application in the detection and treatment of bone-loss diseases, such as osteoporosis, hypercalcemia and bone resorption.
Additionally, antigenic oligopeptides and oligopeptides having the ability to bind to cellular growth factor receptors are derived from the basic peptide structure and the determined TGF polypeptide structures. Finally, compositions and methods, including antibodies raised to the TGF polypeptides and antigenic oligopeptides derived therefrom are provided for use in the health sciences field.
2. Description of the Prior Art
Marquardt and Todaro (1982) J. of Bio. Chem. Vol. 257, No. 9, pp. 5220-5225, (published May 10, 1982) describe the isolation of a low molecular weight human TGF from serum-free medium conditioned by a human metastatic melanoma tumor line by a sequence of process steps including extraction in 1M acetic acid and sequential purification on reversed phase high pressure liquid chromatography eluting first with acetonitrile solvent followed by elution with 1-propanol to afford a purified TGF having characteristic TGF biological activity, e.g., induction of anchorage-independent cell growth. Twardzik et al. (1982) Science 216, pp. 894-897 (published May 21, 1982) report the use of the same purification methodology to purify a TGF from a virus transformed rat cell. The biological activity of the purified materials in the cell culture is also demonstrated. Pike et al. (1982) J. of Bio. Chem. Vol. 257, No. 24, pp. 14628-14631 (published Dec. 25, 1982) disclose that both partially purified rat and human TGF have the ability to activate a protein kinase in human tumor cell membranes and therefore to stimulate phosphorylation of a synthetic tyrosine-containing peptide. The only other molecules so far described that have this activity are EGF, insulin and platelet derived growth factor, all of which are believed to have important physiologic functions in man and animal. Other references of interest are cited in the aforementioned articles.